Evaluation of the Transplantation of Genetically Modified Limbal Epithelial Cells Using a Single Amniotic Membrane Technique.

Abstract

Purpose: Histopathological evaluation of transplanted genetically modified limbal epithelial cells using a single amniotic membrane technique.

Methods: A limbal stem cell deficiency (LSCD) was created in the right eye of normal New Zealand white rabbits (N=16) by performing a 360 degree limbal peritomy and limbalectomy. Intact human amniotic membranes with no cell (N=5), with genetically modified rabbit (N= 4) or human (N=7) limbal epithelial cells (LEC) were secured to the cornea. The LECs were cultured on the stromal surface of the amniotic membrane for 10-14 days prior to transplantation. The cultured cells were transfected with 0.5µg GFP DNA and 4.5µg PAX6 DNA at 4-7 days. The LEC-amniotic membrane (AM) graft was sutured with the cells facing the cornea. Animals with only the AM were euthanized at 45 days, and animals with the genetically modified LECs were euthanized at 130 days post-operatively. The eyes were fixed in Davidson's solution and embedded in paraffin. Four micron sections were stained with Hematoxylin and Eosin. The corneal morphology of the rabbit and human transfected cells were compared to normal rabbit eyes and eyes with only the AM.

Results: The corneas of eyes transplanted with transfected rabbit or human limbal epithelial cells were morphologically similar to normal rabbit corneas, and had 6-7 layers of epithelial cells consisting of normal basal and wing cells.

Conclusions: The transplantation of genetically modified limbal epithelial cells using a single amniotic membrane is effective in functionally reconstructing the corneal epithelium in a LSCD model.

Keywords: 480 cornea: basic science • 637 pathology: experimental • 765 wound healing